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Antimicrobial susceptibility test from apurva sastry

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"Antimicrobial Susceptibility Testing"[MeSH Terms]

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CLSI antimicrobial susceptibility testing disk diffusion broth microdilution standards overview

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https://clsi.org/shop/standards/m07

Antimicrobial Susceptibility Testing (AST)

AST determines the in-vitro response of a bacterium to antimicrobial agents. It helps select an effective drug, detect resistance, guide therapy, and support preparation of an antibiogram.

Terms

  • Susceptible (S): Likely clinical response at usual exposure/dosing.
  • Intermediate / Increased exposure (I): Response may occur when drug exposure is increased or at sites where the drug concentrates.
  • Resistant (R): High likelihood of therapeutic failure despite usual exposure.
  • MIC: Minimum inhibitory concentration, the lowest concentration of an antimicrobial that visibly inhibits bacterial growth.
  • MBC: Minimum bactericidal concentration, the lowest concentration that kills 99.9% of the initial inoculum.
Interpretation must use the latest CLSI or EUCAST organism-drug specific breakpoints, not a single universal zone/MIC cutoff. CLSI M100 also provides QC ranges and guidance on drug reporting and resistance-mechanism confirmation, as summarized in this CLSI standards review.

Indications

Perform AST when:
  1. The organism has variable or predictable acquired resistance, such as E. coli, Klebsiella, Pseudomonas aeruginosa, Acinetobacter, staphylococci, enterococci, and Mycobacterium tuberculosis.
  2. The patient is immunocompromised or has a severe infection.
  3. Resistance may develop rapidly during therapy, for example with P. aeruginosa.
  4. The isolate is from a normally sterile site, such as blood or CSF.
Usually AST is not required when susceptibility is predictable, such as Streptococcus pyogenes to penicillin, or when mixed flora likely represent contamination/colonization. Tietz Textbook of Laboratory Medicine, 7th ed., p. 3282.

Methods of AST

1. Disk diffusion method (Kirby-Bauer method)

Principle

Paper disks containing a fixed concentration of antimicrobial are placed on an agar lawn culture. The drug diffuses outward to form a concentration gradient. If the organism is inhibited, a zone of inhibition appears around the disk.
The zone diameter is measured in millimeters and compared with standard CLSI/EUCAST tables to report S, I, or R.

Procedure

  1. Take 3-5 similar colonies from a pure, fresh culture.
  2. Prepare a suspension equal to 0.5 McFarland turbidity standard.
  3. Inoculate the entire surface of a Mueller-Hinton agar plate evenly with a sterile swab.
  4. Apply antimicrobial disks using sterile forceps or a disk dispenser.
  5. Incubate, commonly at 35 ± 2°C for 16-18 hours for non-fastidious bacteria.
  6. Measure the diameter of the complete inhibition zone in mm.
  7. Interpret with current breakpoint tables.

Advantages

  • Simple, inexpensive, and reproducible.
  • Suitable for routine testing of many non-fastidious bacteria.
  • Multiple drugs can be tested on a single plate.

Limitations

  • Gives categorical result, not an exact MIC.
  • Not suitable for all organisms and drugs.
  • Results are affected by inoculum density, medium composition, agar depth, pH, disk potency, incubation, and reading technique.
  • Not the reference method for most organisms.

Important quality points

  • Use Mueller-Hinton agar of standard composition.
  • Agar depth should be 4 mm. Deep agar can produce false resistance; shallow agar can produce false susceptibility.
  • Use the correct inoculum, incubation atmosphere, temperature, and duration.
  • Test standard quality-control strains, for example E. coli ATCC 25922 and S. aureus ATCC 25923 where appropriate.
Henry's Clinical Diagnosis and Management by Laboratory Methods, p. 1338, notes that agar depth directly changes the diffusion gradient and can produce false susceptibility or resistance.

2. Dilution methods

These methods determine the MIC.

A. Broth macrodilution

  • Serial two-fold dilutions of antimicrobial are prepared in tubes containing broth.
  • A standard bacterial inoculum is added.
  • After incubation, the lowest concentration with no visible growth is the MIC.

B. Broth microdilution

  • Same principle as macrodilution but done in microtiter wells, usually 96-well plates.
  • More economical and suitable for routine or automated systems.
  • Commonly used as a reference method for many aerobic bacteria.

C. Agar dilution

  • Different antimicrobial concentrations are incorporated into agar plates.
  • A standardized bacterial inoculum is spot-inoculated.
  • The lowest concentration preventing visible growth is the MIC.

Advantages

  • Provides a quantitative MIC.
  • Useful for organisms or drugs for which disk diffusion is unreliable.
  • Broth microdilution is widely used for automated systems and is a reference method for many non-fastidious organisms.

Limitations

  • More laborious and costly than disk diffusion when done manually.
  • Requires strict standardization.
  • Agar dilution is difficult for large numbers of isolates.
CLSI M07 describes standardized broth macrodilution, broth microdilution, and agar dilution testing, including inoculum preparation, incubation, interpretation, QC, and limitations. See the CLSI M07 overview.

3. Gradient diffusion method (E-test)

Principle

A plastic strip contains a continuous gradient of antimicrobial concentration. It is placed on an inoculated Mueller-Hinton agar plate.
After incubation, an elliptical zone of inhibition forms. The MIC is read at the point where the ellipse intersects the scale on the strip.

Advantages

  • Easy to perform.
  • Provides an MIC.
  • Useful for fastidious organisms and when testing a small number of isolates.
  • Helpful for drugs such as vancomycin, colistin, and antifungals when an MIC is required, provided the method is validated for that organism-drug pair.

Limitations

  • More expensive than disk diffusion.
  • Results can vary with technique, medium, and reading of trailing endpoints.
  • Not all organism-drug combinations are validated.

4. Automated AST systems

Examples include:
  • VITEK 2
  • Phoenix
  • MicroScan
  • Sensititre

Principle

These systems use broth microdilution or related growth-detection technology. They identify organisms and/or estimate MICs rapidly, then provide S/I/R interpretation.

Advantages

  • Rapid and high-throughput.
  • Less manual work.
  • Standardized reporting and data storage.
  • Useful for hospital laboratory workflow and cumulative antibiograms.

Limitations

  • Expensive.
  • Some unusual resistance phenotypes or unexpected results require confirmation by reference methods.
  • Results must be correlated with organism identification, intrinsic resistance, and clinical context.

5. Molecular methods for resistance detection

These detect genes or mutations associated with resistance.
Examples:
  • mecA/mecC gene or PBP2a detection for MRSA.
  • vanA/vanB for vancomycin-resistant enterococci.
  • Carbapenemase genes such as KPC, NDM, VIM, IMP, OXA-48-like.
  • GeneXpert and PCR-based resistance detection in tuberculosis.

Advantages

  • Rapid.
  • Useful for infection control and early targeted therapy.
  • Can be performed directly on clinical samples in selected settings.

Limitations

  • Detects only known resistance genes/mutations.
  • Presence of a gene may not always equal phenotypic expression.
  • Does not provide a full susceptibility profile.
For staphylococci, cefoxitin resistance is commonly used as a phenotypic surrogate for methicillin resistance; resistance is mediated mainly by mecA, which encodes altered PBP2a. Henry's Clinical Diagnosis and Management by Laboratory Methods, p. 1315.

Factors Affecting AST Results

  1. Inoculum density: Too heavy inoculum may give falsely resistant result.
  2. Age and purity of culture: Use a fresh pure culture.
  3. Medium: Correct Mueller-Hinton medium, pH, cation concentration, and agar depth are necessary.
  4. Disk potency and storage: Disks must be stored correctly and used before expiry.
  5. Incubation conditions: Temperature, duration, atmosphere, and humidity affect results.
  6. Reading method: Measure zones accurately from the back of the plate using reflected light where required.
  7. Quality control: Appropriate ATCC control strains must give acceptable zone or MIC ranges.

Difference between Disk Diffusion and Dilution

FeatureDisk diffusionDilution
ResultZone diameterMIC
OutputS/I/R categoryExact inhibitory concentration plus S/I/R
CostLowHigher
Routine useVery commonCommon in microdilution/automated systems
Reference statusStandard method but generally not reference methodBroth microdilution is reference for many organisms
Best useRoutine screening of non-fastidious bacteriaPrecise MIC, difficult drugs/organisms, automated testing

Short exam answer

Antimicrobial susceptibility testing is an in-vitro method used to determine whether an organism is susceptible, intermediate, or resistant to antimicrobial agents. Main methods are disk diffusion (Kirby-Bauer), dilution methods for MIC determination, gradient diffusion/E-test, automated systems, and molecular detection of resistance genes. Disk diffusion uses antibiotic disks on Mueller-Hinton agar and reports zone diameters. Dilution methods determine MIC and include broth macro/microdilution and agar dilution. Accurate AST requires standardized inoculum, medium, incubation, breakpoint interpretation, and quality-control strains.
Recent reviews found in PubMed focus mainly on AST implementation and antibiogram use rather than changing the basic laboratory principles. One useful systematic review is the 2024 antibiogram review.

Create a one-page AST quick-reference PDF

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mkdir -p "/tmp/workspace/ast-quick-reference/src" "/tmp/workspace/ast-quick-reference/output" && python - <<'PY' from reportlab.lib.pagesizes import A4 from reportlab.pdfgen import canvas from reportlab.lib import colors from reportlab.pdfbase.pdfmetrics import stringWidth from reportlab.pdfbase.ttfonts import TTFont from reportlab.pdfbase import pdfmetrics from reportlab.lib.units import mm from reportlab.pdfbase.pdfmetrics import stringWidth from pypdf import PdfReader out='/tmp/workspace/ast-quick-reference/output/AST_Quick_Reference.pdf' W,H=A4 c=canvas.Canvas(out,pagesize=A4) c.setTitle('Antimicrobial Susceptibility Testing - Quick Reference') c.setAuthor('Orris') # Palette navy=colors.HexColor('#123047'); blue=colors.HexColor('#1B5E7A'); teal=colors.HexColor('#087E8B') light=colors.HexColor('#F2F7F9'); pale=colors.HexColor('#E3F1F3'); line=colors.HexColor('#BCD2D9') ink=colors.HexColor('#162B36'); muted=colors.HexColor('#516B77'); green=colors.HexColor('#177A48'); amber=colors.HexColor('#B36B00'); red=colors.HexColor('#B93636') m=12*mm content_w=W-2*m # helpers def txt(x,y,s,font='Helvetica',size=8,color=ink): c.setFont(font,size); c.setFillColor(color); c.drawString(x,y,s) def rtxt(x,y,s,font='Helvetica',size=8,color=ink): c.setFont(font,size); c.setFillColor(color); c.drawRightString(x,y,s) def wrap(s,font,size,maxw): words=s.split(); lines=[]; cur='' for word in words: test=(cur+' '+word).strip() if stringWidth(test,font,size)<=maxw: cur=test else: lines.append(cur); cur=word if cur: lines.append(cur) return lines def para(x,y,s,width,size=7.4,leading=9.2,color=ink,font='Helvetica'): c.setFont(font,size); c.setFillColor(color) for ln in wrap(s,font,size,width): c.drawString(x,y,ln); y-=leading return y def section(x,y,w,title,accent=teal): c.setFillColor(accent); c.roundRect(x,y-4,w,15,3,fill=1,stroke=0) txt(x+6,y+1,title,'Helvetica-Bold',8.4,colors.white) return y-12 def box(x,y,w,h,fill=light,stroke=line): c.setFillColor(fill); c.setStrokeColor(stroke); c.roundRect(x,y-h,w,h,4,fill=1,stroke=1) def bullet(x,y,label,body,width, color=ink): txt(x,y,u'•','Helvetica-Bold',8.5,teal) txt(x+7,y,label,'Helvetica-Bold',7.5,color) lw=stringWidth(label,'Helvetica-Bold',7.5) return para(x+7+lw+3,y,body,width-lw-10,7.5,9,color) # Header c.setFillColor(navy); c.rect(0,H-37*mm,W,37*mm,fill=1,stroke=0) txt(m,H-16*mm,'ANTIMICROBIAL SUSCEPTIBILITY TESTING','Helvetica-Bold',18,colors.white) txt(m,H-23*mm,'AST QUICK REFERENCE | routine bacteriology laboratory','Helvetica',8.4,colors.HexColor('#D7EDF2')) # small metric cards cards=[('S','Likely response at standard exposure',green),('I','Increased exposure / selected settings',amber),('R','High likelihood of failure',red)] x=W-m-95*mm for k,lab,col in cards: c.setFillColor(colors.white); c.roundRect(x,H-29.5*mm,29*mm,11*mm,3,fill=1,stroke=0) txt(x+4,H-24.7*mm,k,'Helvetica-Bold',10,col) para(x+10,H-22.5*mm,lab,17*mm,5.5,6.3,muted) x+=31.5*mm # Layout colgap=6*mm; cw=(content_w-colgap)/2; lx=m; rx=m+cw+colgap top=H-44*mm # Left column: essentials and methods y=top y=section(lx,y,cw,'1. WHAT AST REPORTS') box(lx,y,cw,43*mm) y-=7 bullet(lx+5,y,'MIC:', 'lowest antimicrobial concentration that prevents visible growth.',cw-10); y-=11 bullet(lx+5,y,'MBC:', 'lowest concentration that kills 99.9% of the starting inoculum.',cw-10); y-=11 bullet(lx+5,y,'Endpoint:', 'usually growth inhibition; interpret every organism-drug pair using current CLSI or EUCAST breakpoints.',cw-10); y-=18 y=section(lx,y,cw,'2. METHODS AT A GLANCE',blue) # table rows=[ ('Disk diffusion','Zone diameter (mm)','Routine, low cost'), ('Broth microdilution','MIC (mg/L)','Reference method for many bacteria'), ('Agar dilution','MIC (mg/L)','Selected drugs / organisms'), ('Gradient strip (E-test)','MIC (mg/L)','Small volume or fastidious isolates'), ('Automated AST','MIC + S/I/R','High-throughput workflow'), ('Molecular assay','Resistance gene / mutation','Rapid targeted detection')] rowh=10.2*mm box(lx,y,cw,7*mm+len(rows)*rowh,colors.white,line) # header c.setFillColor(pale); c.roundRect(lx,y-7*mm,cw,7*mm,4,fill=1,stroke=0) txt(lx+4,y-4.6*mm,'METHOD','Helvetica-Bold',6.5,navy) txt(lx+43*mm,y-4.6*mm,'OUTPUT','Helvetica-Bold',6.5,navy) txt(lx+71*mm,y-4.6*mm,'BEST USE','Helvetica-Bold',6.5,navy) ry=y-7*mm for i,(a,b,d) in enumerate(rows): ry-=rowh if i%2==0: c.setFillColor(light); c.rect(lx+1,ry+1,cw-2,rowh-2,fill=1,stroke=0) txt(lx+4,ry+4.5*mm,a,'Helvetica-Bold',6.6,ink) para(lx+43*mm,ry+5.6*mm,b,24*mm,6.15,7.2,muted) para(lx+71*mm,ry+5.6*mm,d,cw-75*mm,6.15,7.2,muted) y=ry-5*mm y=section(lx,y,cw,'3. KIRBY-BAUER: CORE WORKFLOW') box(lx,y,cw,46*mm,light,line) steps=[ ('1','Fresh pure culture','3-5 similar colonies.'), ('2','Standardize inoculum','0.5 McFarland suspension.'), ('3','Inoculate plate','Confluent lawn on Mueller-Hinton agar.'), ('4','Apply disks','Correct content, spacing, and storage.'), ('5','Incubate','Usually 35 ± 2°C, 16-18 h for non-fastidious bacteria.'), ('6','Measure + interpret','Measure complete zone in mm; apply current breakpoint table.')] sy=y-6*mm for n,head,desc in steps: c.setFillColor(teal); c.circle(lx+8,sy+1.5,3.2,fill=1,stroke=0) c.setFillColor(colors.white); c.setFont('Helvetica-Bold',6); c.drawCentredString(lx+8,sy-.3,n) txt(lx+14,sy,head,'Helvetica-Bold',6.8,ink) para(lx+14+stringWidth(head,'Helvetica-Bold',6.8)+3,sy,desc,cw-27-stringWidth(head,'Helvetica-Bold',6.8),6.65,7.9,muted) sy-=6.4*mm # Right column ry=top ry=section(rx,ry,cw,'4. QUALITY CHECKS THAT CHANGE RESULTS') box(rx,ry,cw,61*mm,colors.white,line) checks=[ ('Inoculum','Too heavy can falsely increase resistance.'), ('Medium','Use validated Mueller-Hinton media. pH and cation content matter.'), ('Agar depth','Target 4 mm. Deep agar can give false resistance; shallow agar can give false susceptibility.'), ('Incubation','Use prescribed time, temperature, and atmosphere.'), ('Disks / strips','Confirm potency, expiry, and correct storage.'), ('QC strains','Run specified ATCC controls; results must fall within accepted ranges.'), ('Unusual results','Confirm with a validated alternative/reference method and assess intrinsic resistance.')] cy=ry-7 for head,desc in checks: txt(rx+5,cy,'✓','Helvetica-Bold',8,green) txt(rx+12,cy,head+':','Helvetica-Bold',7.2,ink) para(rx+12+stringWidth(head+':','Helvetica-Bold',7.2)+3,cy,desc,cw-20-stringWidth(head+':','Helvetica-Bold',7.2),7.1,8.3,muted) cy-=7.6*mm ry-=67*mm ry=section(rx,ry,cw,'5. WHEN TO TEST AND WHEN NOT TO') box(rx,ry,cw,45*mm,light,line) txt(rx+5,ry-7,'TEST when:', 'Helvetica-Bold',7.6,green) ry1=para(rx+5,ry-15,'• resistance is variable or commonly acquired (e.g., Enterobacterales, Pseudomonas, staphylococci, enterococci).\n• infection is severe, invasive, or in an immunocompromised host.\n• resistance may emerge during treatment.',cw-10,7.05,8.6,ink) txt(rx+5,ry-35,'DO NOT routinely test when:', 'Helvetica-Bold',7.6,red) para(rx+5,ry-43,'• susceptibility is predictably uniform (e.g., S. pyogenes to penicillin).\n• mixed growth suggests colonization, contamination, or a poor specimen.',cw-10,7.05,8.6,ink) ry-=51*mm ry=section(rx,ry,cw,'6. INTERPRETATION SAFETY') box(rx,ry,cw,42*mm,colors.white,line) notes=[ ('Never use one universal cutoff.','Breakpoints are specific to the organism, drug, method, indication, and standard used.'), ('Do not equate in-vitro with clinical cure.','Consider source control, site penetration, dose/exposure, host status, and infection severity.'), ('Check intrinsic resistance and selective reporting.','Some drug-organism pairs should not be reported even if an in-vitro value is generated.'), ('Update tables.','CLSI/EUCAST breakpoints and reporting guidance are revised regularly.')] ny=ry-7 for head,desc in notes: txt(rx+5,ny,u'•','Helvetica-Bold',8,blue) txt(rx+12,ny,head,'Helvetica-Bold',7,ink) ny=para(rx+12,ny-8,desc,cw-18,6.8,8,muted)-2 # footer fy=12*mm c.setStrokeColor(line); c.line(m,fy+7*mm,W-m,fy+7*mm) txt(m,fy+2.5*mm,'Source basis: Tietz Textbook of Laboratory Medicine, 7th ed., Ch. 85; Henry\'s Clinical Diagnosis and Management by Laboratory Methods; CLSI M02/M07/M100 concepts.', 'Helvetica',5.9,muted) rtxt(W-m,fy+2.5*mm,'Educational aid - use current local CLSI/EUCAST procedures.', 'Helvetica-Bold',5.9,navy) c.showPage(); c.save() reader=PdfReader(out) assert len(reader.pages)==1, f'Expected 1 page, got {len(reader.pages)}' text=reader.pages[0].extract_text() assert 'ANTIMICROBIAL SUSCEPTIBILITY TESTING' in text and 'KIRBY-BAUER' in text print(out) print('pages',len(reader.pages),'bytes',__import__('os').path.getsize(out),'textchars',len(text)) PY

AST Quick Reference

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